Songguo Xue

Combined 17β-Estradiol with TCDD Promotes M2 Polarization of Macrophages in the Endometriotic Milieu with Aid of the Interaction between Endometrial Stromal Cells and Macrophages

The goal of this study is to elucidate the effects of 17β-estradiol and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) on macrophage phenotypes in the endometriotic milieu. Co-culture of endometrial stromal cells (ESCs) and U937 cells (macrophage cell line) was performed to simulate the endometriotic milieu and to determine the effects of 17β-estradiol and/or TCDD on IL10, IL12 production and HLA-DR, CD86 expression by U937 macrophages. We found that combining 17β-estradiol with TCDD has a synergistic effect on inducing M2 activation when macrophages are co-cultured with ESCs. Moreover, the combination of 17β-estradiol and TCDD significantly enhanced STAT3 and P38 phosphorylation in macrophages. Differentiation of M2 macrophages induced by 17β-estradiol and TCDD were effectively abrogated by STAT3 and P38MAPK inhibitors, but not by ERK1/2 and JNK inhibitors. In conclusion, 17β-estradiol and TCDD in the ectopic milieu may lead to the development of endometriosis by inducing M2 polarization of macrophages through activation of the STAT3 and P38MAPK pathways.

Secretome profile of mouse oocytes after activation using mass spectrum

ObjectiveMammalian oocytes undergo a cortical reaction after fertilization, releasing cortical granules and other proteins into the perivitelline space and inhibiting polyspermy. Few studies have evaluated the biological functions and properties of these proteins.Study designWe investigated mouse oocytes in which the zona pellucida (ZP) was present (ZP-intact group) or absent (ZP-free group).ResultsAfter being activated by Srcl2, secreted proteins are collected from mouse oocytes. Mass spectrometry analysis was performed that identified proteins such as Ldhb, PADi6, Uchl1, Pebp1, Alb, Hsp90aa1, Prss1, trypsinogen 7, trypsin 4, trypsin 10, Sod1, Zp1, Zp2, Zp3, Akap8, Npm2, Pkm2 and Ppia in the ZP-free group. Proteins such as Ldhb, Uchl1, Prss1, trypsin 10, trypsinogen 7, and Ast1 were identified in the ZP-intact groups. The expression of some proteins, including Ldhb, Alb and Sod1, were initially detected following oocyte activation. The finding of four trypsin subtypes, such as Prss1, further support previous observations. Studies investigating the physiological functions and properties of these proteins are ongoing.ConclusionsResearch on these cortical proteins provides a theoretical basis for understanding polyspermy inhibition at the level of ZP and gives technological support for fertilization detection, assessment of oocyte quality and embryonic culture.

New technique for mouse oocyte injection via a modified holding pipette

To improve mouse oocyte survival from intracytoplasmic sperm injection, the sharp tip of the injection pipette has been modified to have a flat end. Here, for the same goal but for a more convenient manipulation, a sharp injection pipette was kept whereas the holding pipette was modified to have a trumpet-shaped opening, which allows deeper injection into the oocyte as it is held. Mouse oocyte injection with mouse and human spermatozoa was performed at 37°C. For the injection of mouse oocyte with mouse sperm head, a significantly higher survival rate (83%) was achieved by utilizing the modified holding pipette than the conventional one (21%; P<0.001) and the fertilization rates were normal and comparable for both methods (82% versus 81%). A superior survival rate (82%) and acceptable normal fertilization rate (71%) were also achieved by utilizing the modified holding pipette for interspecies ICSI (injecting mouse oocyte with human spermatozoon). Taken together, by utilizing a holding pipette with a trumpet-shaped opening, acceptable rates of mouse oocyte survival and fertilization can be achieved using a sharp injection pipette under conditions usual for human oocyte injection.

A novel method for cryopreservation of individual human spermatozoa

The purpose of this study is to develop a novel method for the cryopreservation and efficient post-thaw recovery of individual or small numbers of human spermatozoa. Spermatozoa equilibrated in cryoprotectant buffer were injected with an intracytoplasmic sperm injection (ICSI) needle into a droplet of cryoprotectant on a homemade cryoleaf. The droplet was of cryoprotectant and seminal plasma at a ratio of 1:1. The sperm-loaded cryoleaf was slowly lowered over and stored in liquid nitrogen. Spermatozoa were thawed in a 37°C oil bath without dilution and centrifugation. To test the fertilizing ability of these spermatozoa, the recovered spermatozoa were injected by ICSI into 1-d-old or in vitro-matured human oocytes. Fresh spermatozoa from the same semen samples served as controls. The trials were performed in two separate experiments. In the first set of experiments, 92 spermatozoa were thawed and carefully investigated. The spermatozoa from percutaneous epididymal sperm aspiration had a motility recovery of 92.9% (13/14); ejaculated spermatozoa had a motility recovery of 61.5% (48/78), and only 1.3% (1/78) was lost. Together in the first and second set of experiments, the fertilization rates for the fresh and frozen–thawed spermatozoa were 67.6% (25/37) and 60.6% (40/66), respectively (P = 0.052). The mean embryo cleavage rates in the fresh and frozen–thawed groups were 88% (22/25) and 85% (34/40), respectively (P = 0.990). This cryopreservation method for individual or small numbers of human spermatozoa was efficient and simple. These findings make this method a promising technique for the clinical application of ejaculated sperm from oligozoospermic patients.

Enhancement of the efficiency of oocyte vitrification through regulation of histone deacetylase 6 expression

ObjectiveSuccessful oocyte vitrification (OV) is critical for cryopreservation of the oocytes from female patients with infertility, polycystic ovaries, and gynecologic cancers. Recent evidence suggests that relatively low levels of histone acetylation are critical for maintenance of the maturation capacity of cryopreserved oocytes. However, previous studies have only demonstrated a key role of histone deacetylases (HDAC) 1 and 2 in the cryopreservation of oocytes.MethodsIn this study, we investigated the role of HDAC6 in these settings. We found that mouse oocytes with low HDAC6 levels decreased survival rate, cleavage rate, and blastocyst rate after OV. Bioinformatics analyses were used to predict HDAC6-targeting microRNAs (miRNAs), while the functional binding of miRNAs to HDAC6 mRNA was evaluated by a dual luciferase reporter assay.ResultsAmong all HDAC6-targeting miRNAs, we detected expression of miR-558, miR-527, and miR-762 in mouse oocytes. Specifically, we found that only miR-762 significantly inhibited protein translation of HDAC6 via binding to the 3′-UTR of the HDAC6 mRNA. Transfection of oocytes with HDAC6 or antisense of miR-762 significantly increased the survival rate, the cleavage rate, and blastocyst rate after OV.ConclusionAs a result, our data suggest that induction of HDAC6 levels by miR-762 suppression may improve the current protocol for OV.

Alternations in miRNA Expression in Chronic Stress-Induced Ageing of Leydig Cells

Purpose: Chronic stress represents a critical influence on ageing of Leydig cells in testis. We aimed to investigate expression changes of microRNA (miRNA) associated with stress-induced ageing in Leydig cells.Methods: Brown Norway rats were separated into three groups, young control group, stress group and age group. Physiological changes, including serum corticosterone and testosterone levels, 11β-HSD (11β- hydroxysteroiddehydrogenase) activity, GSH/GSSG (glutathione/glutathione disulfide) ratio and DNA damage after chronic stress treatment were tested by radioimmunoassay and Immunohistochemistry. Differentially expressed miRNAs in Leydig cells between young control group and stress group were identified by miRNA microarray analysis, followed by target prediction of miRNAs. Functional annotation of predicted targets of miRNA was carried out using GeneSpring software and miRNA-target gene interaction network was built by String software.Results: Under chronic stress, increased serum corticosterone levels and reduced testosterone levels were observed. Meanwhile, decline of 11β-HSD activity and GSH/GSSG ratio, increase of DNA damage were also shown in stress group. After performed miRNA microarray analysis, three differentially expressed miRNAs were screened out, including rno-miR-31a-5p, rno-miR-192-5p and rno-miR-191a-3p. Targets of them were mainly involved in apoptosis, cell cycle and transport. In miRNA-target gene interaction network, targets of miR-31a-5p (Tp53, Ywhae and Ndel1), and targets of miR-192-5p (Cdk2, Rac2, Stk3) were with higher degree.Conclusion: These data suggest a central role of miRNA-regulated gene expression in response to chronic stress, especially dys-regulation of rno-miR-31a-5p and rno-miR-192-5p play a vital role in ageing of Leydig cells in testis.